The p53 protein was first detected in a complex with the SV40 large T antigen in rodent cells transformed by simian virus SV40 (Lane, D. P. et al. (1979) Nature, 278:261-263). Subsequently, p53 was shown to be complexed with adenovirus and oncogenic papillomavirus oncoproteins (Sarnow, P. et al. (1982) Cell, 28:387-394; Werness, B. A. et al. (1990) Science, 248:76-79). Initially, p53 protein was considered to be a cellular proto-oncogene but recent observations have indicated that the gene encoding p53 in its native form is a tumor suppressor gene. Experimental support for the role of p53 as a tumor suppressor has been provided by the demonstration that the p53 gene can suppress the growth of transformed murine or human cells and that mutation or deletion of the p53 gene results in loss of this suppressor function (Eliyahn, D. et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86:8763-8767; Baher, S. J. et al. (1990) Science, 249:912-915; Mercer, W. E. et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87:6166-6170). To date, such mutations of the p53 gene have been demonstrated in tumors of the colon, breast, lung, ovary, bladder, and several other organs, making the p53 gene the most commonly mutated gene yet identified in human cancers (Vogelstein, B., (1990) Nature, 348:681-682). Based on the association of tumor progression with alterations in the p53 gene, major research efforts have been devoted to elucidating the potential biological function of p53.
Recent evidence strongly suggests that one function of p53 protein may be in the regulation of gene transcription. Several groups have demonstrated sequence-specific binding of p53 to DNA (Bargonetti et al. (1991) Cell, 65:1083-1091); Kern et al. (1991) Science, 252:1708-1711) and a genomic consensus sequence has been elucidated that consists of two copies of a symmetric 10 base pair (bp) motif separated by 0-13 bp (El-Deiry et al. (1992) Nature Genet., 1:45-49). Placement of this consensus sequence adjacent to a basal promoter linked to chloramphenicol acetyltransferase (CAT) or luciferase reporter genes resulted in induction of the reporter gene when these constructs were cotransfected with a p53 expression vector into mammalian cells (Kern et al. (1992) Science, 256:827-830; Funk W. D. et al. (1992) Mol. Cell. Biol., 12:2866-2871). In addition, the amino-terminus of p53 has been shown to behave as an acidic transcriptional activation domain when fused to GAL4 (Fields, S. et al. (1990) Science, 249:1046-1049).
More recently, wild-type (wt) p53 protein has been shown to directly activate transcription in vitro (Farmer, G. et al. (1992) Nature, 358:83-86). However, despite the experimental evidence supporting a role for p53 protein in transcriptional activation and the high interest in the potential involvement of p53 in tumorigenesis, there are currently only a few methods available for determining the presence of wt or mutant p53 protein in mammalian cells. One widely used method involves laborious DNA sequencing of the p53 gene itself. A major drawback of this approach is that the presence of a normal p53 DNA sequence is not necessarily an accurate predictor of the presence of functional p53 protein in the cells assayed since interference of p53 function by viral proteins or by abnormal binding of p53 protein to endogenous cellular proteins can occur (Momand, J. et al. (1992) Cell, 69:1237-1245; Oliner, J. D. et al. (1992) Nature, 358:80-83). In addition, this approach is both costly and time-consuming.
Another method used for determining the presence of wt or mutant p53 involves the use of antibodies capable of distinguishing between these two forms of p53. However, this approach also has several limitations. First, many of the mutations which arise in the p53 protein are point mutations and not all such mutations would be expected to be distinguished by a limited number of antibodies. Second, since p53 is the most commonly mutated protein identified in human cancers, the number of antibodies necessary to detect all of the different mutant forms of p53 may be quite high; therefore, this method would be impractical and costly. Finally, the use of anti-p53 antibodies to determine the presence of functional p53 in the cell is not an accurate predictor of functional p53 presence for the reasons cited above for the DNA sequencing method. Therefore, while currently used assays can detect the presence of wild-type or mutant p53 protein in mammalian cells, they cannot accurately determine the presence of functional p53 protein in these cells.
One potential approach to developing a method for determining the presence of functional p53 protein in mammalian cells would be to identify a specific gene whose expression is dependent on the presence of functional p53. Recent studies demonstrating a role for p53 protein in the G1 arrest of the cell cycle following damage of DNA by ionizing radiation (Kastan, N. B. et al. (1991) Cancer Res., 51:6304-6311; Kuerbitz, S. J. et al. (1992) Proc. Natl. Acad. Sci. U.S.A., 89:7491-7495). These studies suggested that genes that are differentially regulated after DNA damage and growth arrest may be candidates for p53-inducible genes.
Five gadd (growth-arrest and DNA-damage inducible) genes have been isolated on the basis of induction after DNA-damage in Chinese hamster ovary (CHO) cells. Subsequently, these genes were found to be induced by DNA-damaging agents or other treatments eliciting growth-arrest, such as serum reduction, in a wide variety of mammalian cells (Fornace, A. J. et al. (1989a) Mol. Cell. Biol., 9:4196-4203). In particular, the GADD45 and GADD153 genes have been found to be rapidly and coordinately induced by agents such as methyl methanesulfonate (MMS) that produce high levels of base damage in DNA in every cell line examined, including human, hamster, murine, and rat cells (Fornace, A. J. et al., (1989a); Fornace, A. J. et al. (1992) Ann. NY Acad. Sci., 26:505-524). Recently, the human GADD45 gene was found to be rapidly induced by ionizing radiation (IR) in lymphoblasts and fibroblasts (Papathanasiou, M. A. et al., (1991) Mol. Cell Biol., 11:1009-1016)). This IR response appeared to be distinct from the "gadd" response to MMS and other base-damaging agents because only GADD45 was strongly induced, and induction occurred with doses of IR that produced relatively little DNA base damage. In addition, a recent report (Fornace, A. J. et al. (1991) in Chapman, J. D., Dewey, W. C., Whitmore, G. F. (eds): "In Radiation Research: A Twentieth-Century Perspective", Academic Press, San Diego, p. 213) demonstrated that IR induction of GADD45 is absent in some human tumor cell lines. Taken together, this information suggests a potential role for p53 in the IR response of GADD45.